Microcrystalline diketopiperazine compositions and methods

ABSTRACT

Disclosed herein are DKP microcrystals made by an improved method where they do not irreversibly self-assemble into microparticles. The microcrystals can be dispersed by atomization and re-formed by spray drying into particles having spherical shell morphology. Active agents and excipients can be incorporated into the particles by spray drying a solution containing the components to be incorporated into microcrystalline diketopiperazine particles. In particular, the microcrystalline particle compositions are suitable for pulmonary drug delivery of one or more peptides, proteins, nucleic acids and/or small organic molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US2014/029491, filed Mar. 14, 2014,which claims the benefit of U.S. Provisional Patent Application Ser. No.61/800,520, filed Mar. 15, 2013, the contents of each of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

Disclosed herein are microcrystalline diketopiperazine (DKP) particles,compositions, methods of making the particles and method of using theparticles. In particular, the particles can be used as a delivery systemfor drugs or active agents in the treatment of disease or disorders, forexample, those of endocrine origin, including diabetes and obesity.

BACKGROUND

Delivery of drugs has been a major problem for many years, particularlywhen the compound to be delivered is unstable under the conditionsencountered in the gastro-intestinal tract when administered orally to asubject, prior to reaching its targeted location. For example, it ispreferable in many cases to administer drugs orally, especially in termsof ease of administration, patient compliance, and decreased cost.However, many compounds are ineffective or exhibit low or variablepotency when administered orally. Presumably this is because the drugsare unstable to conditions in the digestive tract or because they areinefficiently absorbed.

Due to the problems associated with oral drug delivery, drug delivery tothe lungs has been explored. For example, typically drugs delivered tothe lungs are designed to have an effect on the tissue of the lungs, forexample, vasodilators, surfactants, chemotherapeutic agents or vaccinesfor flu or other respiratory illnesses. Other drugs, includingnucleotide drugs, have been delivered to the lungs because theyrepresent a tissue particularly appropriate for treatment, for example,for genetic therapy in cystic fibrosis, where retroviral vectorsexpressing a defective adenosine deaminase are administered to thelungs.

Drug delivery to the lungs for agents having systemic effects can alsobe performed. Advantages of the lungs for delivery of systemic agentsinclude the large surface area and the ease of uptake by the lung'smucosal surface. Pulmonary drug delivery systems present manydifficulties, for example, the use of propellants, and aerosolization ofbiological agents such as proteins and peptides can lead todenaturation, and excessive loss of the agent to be delivered. One otherproblem associated with all of these forms of pulmonary drug delivery isthat it is difficult to deliver drugs into the lungs due to problems ingetting the drugs past all of the natural barriers, such as the cilialining the trachea, and in trying to administer a uniform volume andweight of drug.

Accordingly, there is room for improvement in the pulmonary delivery ofdrugs.

SUMMARY

The present disclosure provides improved microcrystalline particles,compositions, methods of making the particles, and methods that allowfor improved delivery of drugs to the lungs for treating diseases anddisorders in a subject. Embodiments disclosed herein achieve improveddelivery by providing crystalline diketopiperazine compositionscomprising microcrystalline diketopiperazine particles having highcapacity for drug adsorption yielding powders having high drug contentof one or more active agents. Powders made with the presentmicrocrystalline particles can deliver increased drug content in lesseramounts of powder dose, which can facilitate drug delivery to a patient.The powders can be made by various methods including, methods utilizingsurfactant-free solutions or solutions comprising surfactants dependingon the starting materials.

Certain embodiments disclosed herein can comprise powders comprising aplurality of substantially uniform, microcrystalline particles, whereinthe particles have a substantially hollow spherical structure andcomprise a shell which can be porous, and comprises crystallites of adiketopiperazine that do not self-assemble.

Certain embodiments disclosed herein comprises powders comprising aplurality of substantially uniform, microcrystalline particles, whereinthe particles have a substantially hollow spherical structure andcomprise a shell which can be porous, and comprises crystallites of adiketopiperazine that do not self-assemble, and the particles have avolumetric median geometric diameter less than 5 μm.

In a particular embodiment herein, up to about 92% of themicrocrystalline particles have a volumetric median geometric diameterof≤5.8 μm. In one embodiment, the particle's shell is constructed frominterlocking diketopiperazine crystals having one or more drugs adsorbedon their surfaces. In some embodiments, the particles can entrap thedrug in their interior void volume and/or combinations of the drugadsorbed to the crystallites' surface and drug entrapped in the interiorvoid volume of the spheres.

In certain embodiments, a diketopiperazine composition comprising aplurality of substantially uniformly formed, microcrystalline particlesis provided, wherein the particles have a substantially hollow sphericalstructure and comprise a shell comprising crystallites of adiketopiperazine that do not self-assemble; wherein the particles areformed by a method comprising the step of combining diketopiperazinehaving a trans isomer content ranging from about 45% to 65% in asolution and a solution of acetic acid without the presence of asurfactant and concurrently homogenizing in a high shear mixer at highpressures of up to 2,000 psi to form a precipitate; washing theprecipitate in suspension with deionized water; concentrating thesuspension and drying the suspension in a spray drying apparatus.

The method can further comprise the steps of adding with mixing asolution comprising an active agent or an active ingredient such as adrug or bioactive agent prior to the spray drying step so that theactive agent or active ingredient is adsorbed and/or entrapped on orwithin the particles. Particles made by this process can be in thesubmicron size range prior to spray-drying.

In certain embodiments, a diketopiperazine composition comprising aplurality of substantially uniformly formed, microcrystalline particlesis provided, wherein the particles have a substantially hollow sphericalstructure and comprise a shell comprising crystallites of adiketopiperazine that do not self-assemble, and the particles have avolumetric mean geometric diameter less than equal to 5 μm; wherein theparticles are formed by a method comprising the step of combiningdiketopiperazine in a solution and a solution of acetic acid without thepresence of a surfactant and concurrently homogenizing in a high shearmixer at high pressures of up to 2,000 psi to form a precipitate;washing the precipitate in suspension with deionized water;concentrating the suspension and drying the suspension in a spray dryingapparatus.

The method can further comprise the steps of adding with mixing asolution comprising an active agent or an active ingredient such as adrug or bioactive agent prior to the spray drying step so that theactive agent or active ingredient is adsorbed and/or entrapped on orwithin the particles. Particles made by this process can be in thesubmicron size range prior to spray-drying.

In certain embodiments, a diketopiperazine composition comprising aplurality of substantially uniformly formed, microcrystalline particlesis provided, wherein the particles have a substantially hollow sphericalstructure and comprise a shell comprising crystallites of adiketopiperazine that do not self-assemble, and the particles have avolumetric mean geometric diameter less than equal to 5 μm; wherein theparticles are formed by a method comprising the step of combiningdiketopiperazine in a solution and a solution of acetic acid without thepresence of a surfactant and without the presence of an active agent,and concurrently homogenizing in a high shear mixer at high pressures ofup to 2,000 psi to form a precipitate; washing the precipitate insuspension with deionized water; concentrating the suspension and dryingthe suspension in a spray drying apparatus.

The method can further comprise the steps of adding with mixing asolution comprising an active agent or an active ingredient such as adrug or bioactive agent prior to the spray drying step so that theactive agent or active ingredient is adsorbed and/or entrapped on orwithin the particles. Particles made by this process can be in thesubmicron size range prior to spray-drying.

In one embodiment, the composition can comprise microcrystallineparticles comprising one or more active ingredients; wherein the activeingredients are peptides, proteins, nucleic acid molecules, smallorganic molecules, or combinations thereof. In embodiments wherein theactive ingredient is a peptide, oligopeptide, polypeptide or protein,the peptide, oligopeptide, polypeptide or protein can be an endocrinehormone, a neurotransmitter, a vasoactive peptide, a receptor peptide, areceptor agonist or antagonist, and the like. In some embodiments, theendocrine hormone is insulin, parathyroid hormone, calcitonin, glucagon,glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, or an analogof said endocrine hormone. In embodiments, excipients can beincorporated into the particles by addition to one, another, or allfeedstocks used in the spray drying step.

In one embodiment wherein the composition comprises insulin as theactive ingredient, the compositions can contain insulin in amount up to,for example, 9 units or 10 units per milligram of powder to be deliveredto a patient. In this embodiment, insulin can be delivered to a patientin amounts up to, for example, 100 units in a single inhalation using adry powder inhaler. The compositions can be administered to a patient inneed of insulin for the treatment of diabetes and/or hyperglycemia.

In an exemplary embodiment, the crystalline diketopiperazine compositioncomprises a diketopiperazine of the formula2,5-diketo-3,6-bis(N-X-4-aminoalkyl)piperazine, wherein alkyl denotes analkyl containing 3 to 20 carbon atoms, including propyl, butyl, pentyl,hexyl, heptyl and the like; and the formula is, for example,2,5-diketo-3,6-bis(N-X-4-aminobutyl)piperazine, wherein X is selectedfrom the group consisting of fumaryl, succinyl, maleyl, malonyl, andglutaryl, or a salt thereof. In a particular embodiment, thediketopiperazine is(3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine having theformula:

In various embodiments, a method of making dry powders comprisingmicrocrystalline particles suitable for pulmonary administration isprovided; wherein the method can be carried out using surfactant-freesolutions, or solutions comprising a surfactant. In one aspect thediketopiperazine comprises a trans isomer content ranging from about 45%to 65%.

Certain embodiments disclosed herein include methods of producing drypowders comprising crystalline diketopiperazine microparticles fromstarting materials comprising free acid diketopiperazines.

Certain embodiments disclosed herein include methods of producing drypowders comprising crystalline diketopiperazine microparticles fromstarting materials comprising diketopiperazine salts.

In one embodiment, the method comprises:

-   -   dissolving a diketopiperazine in aqueous ammonia to form a first        solution;    -   feeding the first solution and a second solution comprising        about 10.5% acetic acid concurrently to a high shear mixer at an        approximate pH of less than 6.0 under high pressure;    -   homogenizing the first solution and second solution to form a        suspension comprising crystallites of the diketopiperazine in        the suspension, wherein the suspension has a bimodal        distribution of crystallites having particle sizes ranging from        about 0.05 μm to about 10 μm in diameter;    -   atomizing the suspension under an air or gas stream; and    -   reforming particles by spray-drying into a dry powder comprising        the microcrystalline particles having substantially hollow        spheres.

In another embodiment, the method comprises:

-   -   dissolving a diketopiperazine in aqueous sodium hydroxide and        optionally a surfactant to form a first solution;    -   feeding the first solution and a second solution comprising        about 10.5% acetic acid, and optionally a surfactant,        concurrently to a high shear mixer at an approximate pH of less        than 6.0 under high pressure;    -   homogenizing the first solution and second solution to form a        suspension comprising crystallites of the diketopiperazine in        the suspension, wherein the suspension has a bimodal        distribution of crystallites having particle sizes ranging from        about 0.05 μm to about 10 μm in diameter and comprising a trans        isomer content ranging from about 45% to 65%;    -   atomizing the suspension under an air or gas stream; and    -   reforming particles by spray-drying into a dry powder comprising        the microcrystalline particles having substantially hollow        spheres.

In one embodiment, the method comprises:

-   -   dissolving a diketopiperazine in aqueous ammonia to form a first        solution;    -   feeding the first solution and a second solution comprising        about 10.5% acetic acid concurrently to a high shear mixer at an        approximate pH of less than 6.0 under high pressure to form a        suspension comprising crystallites of the diketopiperazine in        the suspension, wherein the suspension has a bimodal        distribution of crystallites having particle sizes ranging from        about 0.05 μm to about 10 μm in diameter;    -   atomizing the suspension under an air or gas stream; and    -   reforming particles by spray-drying into a dry powder comprising        the microcrystalline particles having substantially hollow        spheres.

The method can further comprise the step of adding a third solution tothe diketopiperazine crystallite suspension prior to atomizing thesuspension; wherein the solution contains a drug or a pharmaceuticallyactive ingredient, and the atomizing step can be performed using anexternal mixing 2-fluid nozzle into a spray dryer fitted with a highefficiency cyclone separator under air or gas, including nitrogen gas.

In certain embodiments, the particles in suspension have a particle sizedistribution as a bimodal curve as measured by laser diffraction;wherein a first peak of particles has an average particle size of about0.2 μm to about 0.4 μm, and a second peak of particles having an averagesize of about 2.1 μm to about 2.4 μm in diameter.

In some embodiments, the step of atomizing the suspension uses anitrogen stream of about 700 liters of nitrogen per hour as the processgas, and the nozzle temperature can be kept at about 25° C.

Microcrystalline particles formed by the method above do notself-assemble when suspended in a solution, such as water or otheraqueous-based solvent. In a particular embodiment, the method comprisesa diketopiperazine of the formula2,5-diketo-3,6-bis(N-X-4-aminobutyl)piperazine, wherein X is selectedfrom the group consisting of fumaryl, succinyl, maleyl, malonyl, andglutaryl. In a specific embodiment, the method comprises homogenizing ina high shear mixer a solution of a diketopiperazine, wherein thediketopiperazine is(bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine, or a saltthereof, including, disodium, dipotassium, magnesium, calcium, anddilithium salts.

In an embodiment, a crystalline diketopiperazine composition comprisinga plurality of microcrystalline particles substantially uniform in sizeis obtained as a product of the spray drying step.

In an embodiment, a crystalline diketopiperazine composition comprisinga plurality of microcrystalline particles with a bimodal sizedistribution is obtained as a product of the crystallite formation step.

When a disruption step is used, the larger species of the bimodaldistribution can be shifted to smaller sizes.

Certain embodiments comprise a method of forming microcrystallineparticles of a diketopiperazine acid for making dry powders carryinglarge drug content comprises using a salt of a diketopiperazine as astarting compound, including,2,5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine disodium salt, themethod comprises:

-   -   dissolving a diketopiperazine salt in water comprising a        surfactant in an amount from about 0.2% to about 6% (w/w) to        form a first solution;    -   combining the first solution with a second solution comprising        from about 8% to about 12% (w/w) acetic acid concurrently in a        high shear mixer at an approximate pH of less than 6.0 under        high pressure;    -   homogenizing the first solution and second solution to form a        suspension comprising crystallites of the diketopiperazine in        the suspension, wherein the suspension has a bimodal        distribution of crystallites having particle sizes ranging from        about 0.05 μm to about 10 μm in diameter;    -   atomizing the suspension under an air or gas stream; and    -   reforming the particles by spray-drying into a dry powder        comprising microcrystalline particles of the diketopiperazine        acid having substantially hollow spheres.

In a specific embodiment, microcrystalline particles can be made by aprocess comprising, preparing a first solution comprising adiketopiperazine in water, for example,2,5-diketo-3,6-(N-fumaryl-4-aminobutyl)piperazine disodium salt and asurfactant such as polysorbate 80; preparing a second solutioncomprising acetic acid at a concentration about 10.5% (w/w) and asurfactant at a concentration of about 0.5% (w/w); mixing the firstsolution and the second solution in a high shear mixer to form asuspension; optionally testing the suspension to determine the particlesize distribution so that the suspension comprises a bimodal particlesize distribution, with particles ranging in size from about 0.2 μm toabout 10 μm in diameter, wherein a first peak of particles have anaverage diameter of about 0.4 μm and a second peak of particle have anmean diameter of about 2.4 μm, and spray-drying the suspension to obtaina dry powder.

Certain embodiments can comprise a disruption step to reduce the size ofthe larger-sized population in the bimodal distribution, for exampleutilizing sonication, stirring, or homogenization. In embodiments thedisruption step can be performed prior to atomizing the suspension.

In embodiments herein, the method for making the microcrystallinediketopiperazine particles can further include a wash step usingdeionized water. In one embodiment, the atomizing step can be performed,for example, using an external mixing 2-fluid nozzle into a spray dryerfitted with a high efficiency cyclone separator.

The method can further comprise the step of adding a solution comprisingone or more active agents to the suspension prior to dispersing and/orspray drying, wherein the active agent is a peptide, an oligopeptide, apolypeptide, a protein, a nucleic acid molecule, or a small organicmolecule. The peptides can be endocrine hormones, including insulin,parathyroid hormone, calcitonin, glucagon, glucagon-like peptide 1,oxyntomodulin, peptide YY, leptin, or an analog of said endocrinehormone and the like. The method can optionally comprise the step ofadding a solution comprising a surfactant, and/or a pharmaceuticallyacceptable carrier, including amino acids such as leucine, isoleucine,and/or monosaccharides, disaccharides, or oligosaccharides, such aslactose, trehalose, and the like, or sugar alcohols, including,mannitol, sorbitol, and the like.

In another embodiment, a composition comprising more than one activeagent can be made using the present method. The method of making suchcomposition comprises the steps of making microcrystallinediketopiperazine particles comprising more than one active agent whereineach active agent/ingredient is processed separately in a solution andadded to separate suspensions of diketopiperazine particles and solutionconditions are changed to promote adsorption of the active agent ontothe surfaces of the crystallites, then the two or more separatesuspensions comprising the active agents are blended prior to dispersingand spray-drying the particles. In a variant procedure, the blendincludes a suspension containing diketopiperazine particles withoutactive agent, for example in order to achieve a lower overall content ofthe active agent. In an alternate embodiment, the one or moreindependent solutions containing a single active agent can be combinedwith a single suspension comprising the diketopiperazine particles priorto dispersing and spray-drying the particles. The resultant dry powdercomprises a composition comprising two or more active ingredients. Inthese embodiments, the amount of each ingredient in the composition canbe controlled depending on the need of the patient population to betreated.

In another embodiment, the dry powder comprises a composition comprising2,5-diketo-3,6-bis(N-X-4-aminobutyl)piperazine, wherein X is fumaryl andthe composition comprises substantially homogeneous microcrystallineparticles comprising a drug; wherein the particles are substantiallyspherical in shape having a substantially hollow core and thecrystallites form a shell of the sphere. In another embodiment, the drypowders comprise a diketopiperazine of the formula2,5-diketo-3,6-bis(N-X-4-aminobutyl)piperazine and a drug, wherein thedrug is a peptide, wherein the peptide can be of various peptidelengths, molecular sizes or masses, including; insulin, glucagon-likepeptide-1, glucagon, exendin, parathyroid hormone, calcitonin,oxyntomodulin, and the like.

Further embodiments include drug delivery systems comprising an inhalerwith or without a cartridge, wherein the cartridge is a unit dose drypowder medicament container, for example, a cartridge, and a powdercomprising the particles disclosed herein and an active agent. In oneembodiment, the delivery system for use with the dry powders includes aninhalation system comprising a high resistance inhaler having airconduits which impart a high resistance to airflow through the conduitsfor deagglomerating and dispensing the powder. In one embodiment, theinhalation system has a resistance value of, for example, approximately0.065 to about 0.200 (√kPa)/liter per minute. In certain embodiments,the dry powders can be delivered effectively by inhalation with aninhalation system wherein the peak inhalation pressure differential canrange from about 2 to about 20 kPa, which can produce resultant peakflow rates of about between 7 and 70 liters per minute. In certainembodiments, the inhalation system is configured to provide a singledose by discharging powder from the inhaler as a continuous flow, or asone or more pulses of powder delivered to a patient. In some embodimentsdisclosed herewith, the dry powder inhaler system comprises apredetermined mass flow balance within the inhaler, wherein the inhalerconduits are designed to have varied flow distribution during aninhalation. For example, a flow balance of approximately 10% to 70% ofthe total flow exiting the inhaler and into the patient is delivered byone or more dispensing ports, which airflow passes through an airconduit designed with an area for containing a powder formulation, andwherein approximately 30% to 90% air flow is generated from otherconduits of the inhaler during an inhalation maneuver. Moreover, bypassflow, or flow not entering and exiting the area of powder containmentsuch as through a cartridge, can recombine with the flow exiting thepowder dispensing port within the inhaler to dilute, accelerate andultimately deagglomerate the fluidized powder prior to exiting themouthpiece. In one embodiment, flow rates ranging from about 7 to 70liters per minute result in greater than 75% of the container or thecartridge contents dispensed in fill masses between 1 and 50 mg. Incertain embodiments, an inhalation system as described above can emit arespirable fraction/fill of a powder dose at percentages greater than40% in a single inhalation, greater than 50%, greater than 60%, orgreater than 70%.

In certain embodiments, drug delivery systems comprising inhalers cancomprise inhalers particularly suited for use with the morphology of theparticles comprising the dry powder, such as for example a crystallineor amorphous morphology.

In particular embodiments, an inhalation system is provided comprising adry powder inhaler, a dry powder formulation comprising microcrystallineparticles of fumaryl diketopiperazine having an FDKP trans isomercontent between 45% and 65% and one or more than one active agents. Insome aspects of this embodiment of the inhalation system, the dry powderformulation is provided in a unit dose cartridge. Alternatively, the drypowder formulation can be preloaded in the inhaler. In this embodiment,the structural configuration of the inhalation system allows thedeagglomeration mechanism of the inhaler to produce respirable fractionsgreater than 50%; that is, more than half of the powder contained in theinhaler (cartridge) is emitted as particles of less than 5.8 μm. Theinhalers can discharge greater than 85% of a powder medicament containedwithin a container during dosing. In certain embodiments, the inhalerscan discharge greater than 85% of a powder medicament contained in asingle inhalation. In one embodiment, the inhalers can discharge greaterthat 90% of the cartridge contents or container contents in less than 3seconds at pressure differentials between 2 and 5 kPa with fill massesranging up to 30 mg.

Embodiments disclosed herein also include methods. In one embodiment, amethod of treating an endocrine-related disease or disorder comprisingadministering to a person in need thereof a dry powder formulationcomprising FDKP microcrystalline particles comprising an FDKP that canhave a trans isomer content of about 45 to about 65% and a drug suitableto treat said disease or disorder wherein the microparticles areproduced by the present methods. One embodiment includes a method oftreating an insulin-related disorder comprising administering a drypowder comprising microcrystalline particles of FDKP described above toa person in need thereof. The method comprises administering to asubject a dry powder formulation comprising microcrystalline particlesof fumaryl diketopiperazine having a trans isomer content ranging fromabout 45% to 65%, which are particles are hollow spheres and do notcontain any surfactant. In various embodiments an insulin-relateddisorder can specifically include or exclude any or all of pre-diabetes,type 1 diabetes mellitus (honeymoon phase, post-honeymoon phase, orboth), type 2 diabetes mellitus, gestational diabetes, hypoglycemia,hyperglycemia, insulin resistance, secretory dysfunction, impairedearly-phase release of insulin, loss of pancreatic β-cell function, lossof pancreatic β-cells, and metabolic disorder. In one embodiment, thedry powder comprises insulin. In other embodiments, the dry powdercomprises oxyntomodulin, peptide YY, leptin, oxytocin, glucagon, anexendin, GLP-1 analogs thereof or combinations thereof.

Another embodiment disclosed herein includes a method of delivering apeptide including, GLP-1, oxyntomodulin, peptide YY, oxytocin, insulinto a patient in need thereof comprising administering a dry powdercomprising diketopiperazine microcrystalline particles disclosed hereinto the deep lung by inhalation of the dry powder by the patient. Inaspects of this embodiment, particular features of an inhaler system arespecified.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the examplesdisclosed herein. The disclosure may be better understood by referenceto one or more of these drawings in combination with the detaileddescription of specific embodiments presented herein.

FIGS. 1A and 1B are a scanning electron micrographs (SEM) of fumaryldiketopiperazine particles comprising insulin and showing the solidcompositions of the particles lyophilized at low (1A) and highmagnification (1B).

FIG. 2 depicts a graphic representation of the particle sizedistribution of the particles depicted in FIGS. 1A and 1B as measured bythe probability density function (pdf, left y-axis) and cumulativedistribution function (cdf, right y-axis) scale.

FIG. 3 depicts a graphic representation of the particle sizedistribution of particles obtained from an embodiment prepared from asuspension wherein the microcrystalline particles are formed withoutsurfactant in any of the solutions used. The graph shows a typicalbimodal distribution of the microcrystalline particles as measured bythe probability density function (pdf, left y-axis) scale and cumulativedistribution function (cdf, right y-axis) scale.

FIG. 4 depicts an SEM at low magnification (2500×) of FDKP particlesrecovered from an embodiment herein wherein a surfactant-free particlesuspension is lyophilized.

FIG. 5 depicts a graphic representation of the lyophilized particle sizedistribution in suspension as depicted in FIG. 4 formed withoutsurfactant and showing an increase in particle size as measured by theprobability density function (pdf, left y-axis) scale and cumulativedistribution function (cdf, right y-axis) scale.

FIG. 6 depicts an SEM (at 2500×) of a claimed embodiment showingmicrocrystalline particles made from surfactant-free solutions whichwere spray-dried.

FIG. 7 depicts a graphic representation of particle size distribution ofspray-dried surfactant-free particles dispersed in water.

FIG. 8 depicts a graphic representation of the particle sizedistribution of spray-dried surfactant-free particles dispersed in 0.01M HCl (pH 2).

FIG. 9 depicts a graphic representation of the bimodal particle sizedistribution of a suspension formed by crystallizing Na₂FDKP with aceticacid in the presence of surfactant.

FIGS. 10A and 10B depict two scanning electron micrographs of particlesprepared by spray drying the suspension of crystals prepared fromNa₂FDKP at 2,500× (10A) and 10,000× (10B) magnifications.

FIGS. 11A and 11B depict scanning electron micrographs of spray-driedsurfactant-free FDKP particles with approximately 10 wt % insulin at2,500× (11A) and at 5,000× (11B) magnifications.

FIGS. 12A and 12B are two scanning electron micrographs of particlesprepared by spray drying the suspension of crystals prepared from Na₂DKPat 2,500× (12A) and at 10,000× (12B) magnification.

FIG. 13 depicts a graphic representation of the size distribution ofparticles formed by spray drying a suspension of FDKP crystallized froma solution of Na₂FDKP and polysorbate 80. The particles were dispersedin water for the measurement.

FIG. 14 depicts a graphic representation of the particle sizedistributions of a spray-dried combination powder and the crystallitesuspension with the individual active agents. The number 1 representsthe particle size distribution of the combined microcrystalline powdercomposition comprising two different active agents; in separatediketopiperazine-active agent particle suspension, wherein onecomposition contained particles of FDKP-GLP-1 and the other containedFDKP-insulin (3) in suspensions which were combined prior to beingspray-dried.

DETAILED DESCRIPTION

As stated, drug delivery to the lungs offers many advantages. It isdifficult to deliver drugs into the lungs, however, due to problems intransporting the drugs past natural physical barriers in a uniformvolume and weight of the drug. Disclosed herein are crystallinediketopiperazine compositions, dry powders and methods of making theparticles. The crystalline composition and dry powder therefrom comprisediketopiperazines microcrystalline particles, which are substantiallyuniformly defined spheres comprising a shell comprising crystallites ofthe diketopiperazine and a core. In certain embodiments the core can behollow. In one embodiment, the diketopiperazine has a defined transisomer content which can be beneficial to the particles as drug deliveryagents, methods of making the particles and methods of treatment usingthe particles. The particles disclosed herein have higher capacity forcarrying and delivering drug content to the patient in smaller dosesthan standard, prior art particles.

As used herein, an “analog” includes compounds having structuralsimilarity to another compound. Thus, compounds having structuralsimilarity to another (a parent compound) that mimic the biological orchemical activity of the parent compound are analogs. There are nominimum or maximum numbers of elemental or functional groupsubstitutions required to qualify a compound as an analog provided theanalog is capable of mimicking, in some relevant fashion, eitheridentically, complementarily or competitively, with the biological orchemical properties of the parent compound. In some instances an analogcomprises a fragment of the parent compound either in isolation orlinked to another molecule and may contain other alterations as well.Analogs of the compounds disclosed herein may have equal, lesser orgreater activity than their parent compounds.

As used herein, the term “microparticle” refers to a particle with adiameter of about 0.5 to about 1000 μm, irrespective of the preciseexterior or interior structure. Microparticles having a diameter ofbetween about 0.5 and about 10 microns can reach the lungs, successfullypassing most of the natural barriers. A diameter of less than about 10microns is required to navigate the turn of the throat and a diameter ofabout 0.5 microns or greater is required to avoid being exhaled. Toreach the deep lung (or alveolar region) where most efficient absorptionis believed to occur, it is preferred to maximize the proportion ofparticles contained in the “respirable fraction” (RF), generallyaccepted to be those particles with an aerodynamic diameter of about 0.5to about 5.7 microns, though some references use somewhat differentranges, as measured using standard techniques, for example, with anAndersen Cascade Impactor. Other impactors can be used to measureaerodynamic particle size such as the NEXT GENERATION IMPACTOR™ (NGI™,MSP Corporation), for which the respirable fraction is defined bysimilar aerodynamic size, for example <6.4 μm. In some embodiments, alaser diffraction apparatus is used to determine particle size, forexample, the laser diffraction apparatus disclosed in U.S. patentapplication Ser. No. 12/727,179, filed on Mar. 18, 2010, which isincorporated herein in its entirety for its relevant teachings, whereinthe volumetric median geometric diameter (VMGD) of the particles ismeasured to assess performance of the inhalation system. For example, invarious embodiments cartridge emptying of ≥80%, 85%, or 90% and a VMGDof the emitted particles of ≤12.5 μm, ≤7.0 μm, ≤5.8 μm or ≤4.8 μm canindicate progressively better aerodynamic performance. Embodimentsdisclosed herein show that FDKP particles with a trans isomer content ofbetween about 45% to about 65% exhibit characteristics beneficial todelivery of drugs to the lungs such as improved aerodynamic performance.

Respirable fraction on fill (RF/fill) represents the % of powder in adose that is emitted from an inhaler upon discharge of the powdercontent filled for use as the dose, and that is suitable forrespiration, i.e., the percent of particles from the filled dose thatare emitted with sizes suitable for pulmonary delivery, which is ameasure of particle aerodynamic performance. As described herein, aRF/fill value of 40% or greater than 40% reflects acceptable aerodynamicperformance characteristics. In certain embodiments disclosed herein,the respirable fraction on fill can be greater than 50%. In an exemplaryembodiment, a respirable fraction on fill can be up to about 80%,wherein about 80% of the fill is emitted with particle sizes <5.8 μm asmeasured using standard techniques.

As used herein, the term “dry powder” refers to a fine particulatecomposition that is not suspended or dissolved in a propellant, carrier,or other liquid. It is not meant to necessarily imply a complete absenceof all water molecules.

Specific RF/fill values can depend on the inhaler used to deliver thepowder. Powders generally tend to agglomerate and certain crystallineDKP particles form particularly cohesive powders. One of the functionsof a dry powder inhaler is to deagglomerate the powder so that theresultant particles comprise a respirable fraction suitable fordelivering a dose by inhalation. However, deagglomeration of cohesivepowders is typically incomplete so that the particle size distributionseen when measuring the respirable fraction as delivered by an inhalerwill not match the size distribution of the primary particles, that is,the profile will be shifted toward larger particles. Inhaler designsvary in their efficiency of deagglomeration and thus the absolute valueof RF/fill observed using different designs will also vary. However,optimal RF/fill as a function of isomeric content will be the same frominhaler to inhaler.

As used herein, the term “about” is used to indicate that a valueincludes the standard deviation of the measurement for the device ormethod being employed to determine the value.

As used herein, the term “surfactant-free” is used to indicate that nosurfactant was present in any of the reagents, including, solutions,and/or suspensions used in the process of making the microcrystallineparticles.

As used herein, the term “crystallite” is used to refer to the integralcrystalline units of a diketopiperazine particle, which can have varyingsizes.

As used herein, “microcrystalline particles” comprise crystallites of adiketopiperazineand have a particle size distribution of from 0.05 μm toabout 100 μm, as measured by laser diffraction having particles sizes ofless than 50 μm, less than 20 μm, or less than 10 μm in diameter. In anembodiment the crystallites can range in size from 0.01 to 1 μm.

Diketopiperazines

One class of drug delivery agents that has been used to overcomeproblems in the pharmaceutical arts such as drug instability and/or poorabsorption are the 2,5-diketopiperazines. 2,5-Diketopiperazines arerepresented by the compound of the general Formula 1 as shown belowwherein E₁ and E₂ are independently N or more particularly NH. In otherembodiments, E₁ and/or E₂ are independently an oxygen or a nitrogen sothat wherein either one of the substituents for E₁ and E₂ is an oxygenand the other is a nitrogen the formula yields the substitution analogdiketomorpholine, or when both E₁ and E₂ are oxygen the formula yieldsthe substitution analog diketodioxane.

These 2,5-diketopiperazines have been shown to be useful in drugdelivery, particularly those bearing acidic R₁ and R₂ groups asdescribed in, for example, U.S. Pat. No. 5,352,461 entitled “SelfAssembling Diketopiperazine Drug Delivery System;” U.S. Pat. No.5,503,852 entitled “Method For Making Self-Assembling DiketopiperazineDrug Delivery System;” U.S. Pat. No. 6,071,497 entitled “MicroparticlesFor Lung Delivery Comprising Diketopiperazine;” and 6,331,318 entitled“Carbon-Substituted Diketopiperazine Delivery System,” each of which isincorporated herein by reference in its entirety for all that it teachesregarding diketopiperazines and diketopiperazine-mediated drug delivery.Diketopiperazines can be formed into microparticles that incorporate adrug or microparticles onto which a drug can be adsorbed. Thecombination of a drug and a diketopiperazine can impart improved drugstability and/or absorption characteristics. These microparticles can beadministered by various routes of administration. As dry powders themicroparticles can be delivered by inhalation to specific areas of therespiratory system, including the lungs.

Such prior art microparticles are typically obtained by pH-basedprecipitation of the free acid (or base) resulting in self-assembledmicroparticles comprised of aggregated crystalline plates with a roseatemorphology. The stability of the particle can be enhanced by smallamounts of a surfactant, such as polysorbate-80, in the DKP solutionfrom which the particles are precipitated (see for example U.S. Pat. No.7,799,344, entitled “Method of drug formulation based on increasing theaffinity of crystalline microparticle surfaces for active agents” whichis incorporated herein by reference in its entirety for all that itteaches regarding the formation and loading of DKP microparticles anddry powders thereof). Ultimately solvent can be removed to obtain a drypowder. Methods of solvent removal include lyophilization and spraydrying (see for example U.S. Pat. No. 8,039,431 entitled “A method forimproving the pharmaceutic properties of microparticles comprisingdiketopiperazine and an active agent” and U.S. Pat. No. 6,444,226entitled “Purification and stabilization of peptide and proteinpharmaceutical agents” each of which is incorporated herein by referencein its entirety for all that it teaches regarding the formation andloading of DKP microparticles and dry powders thereof). The particlesdisclosed herein are distinct from the prior art particles in that theyare physically, and morphologically distinct entities and are made by animproved method. The present disclosure refers to FDKP to be understoodas the free acid or the dissolved anion.

Other prior art particles are obtained by spray drying DKP solutions toobtain particles of the amorphous DKP salts typically with acollapsed-spherical morphology such as those disclosed in U.S. Pat. Nos.7,820,676 and 8,278,308, entitled “Diketopiperazine salts for drugdelivery and related methods.”

Methods for synthesizing diketopiperazines are described in, forexample, Katchalski, et al., J. Amer. Chem. Soc. 68, 879-880 (1946) andKopple, et al., J. Org. Chem. 33(2), 862-864 (1968), the teachings ofwhich are incorporated herein by reference in their entirety.2,5-Diketo-3,6-di(aminobutyl)piperazine (Katchalski et al. refer to thisas lysine anhydride) can also be prepared via cyclodimerization ofN-ε-P-L-lysine in molten phenol, similar to the Kopple method, followedby removal of the blocking (P)-groups with an appropriate reagent andconditions. For example, CBz-protecting groups can be removed using 4.3M HBr in acetic acid. This route uses a commercially available startingmaterial, it involves reaction conditions that are reported to preservestereochemistry of the starting materials in the product and all stepscan be easily scaled up for manufacture. Methods for synthesizingdiketopiperazines are also described in U.S. Pat. No. 7,709,639,entitled, “Catalysis of Diketopiperazine Synthesis,” which is alsoincorporated by reference herein for its teachings regarding the same.

Fumaryl diketopiperazine(bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketo-diketopiperazine; FDKP) isone preferred diketopiperazine for pulmonary applications:

FDKP provides a beneficial microparticle matrix because it has lowsolubility in acid but is readily soluble at neutral or basic pH. Theseproperties allow FDKP to crystallize and the crystals to self-assembleinto microparticles under acidic conditions. The particles dissolvereadily under physiological conditions where the pH is neutral. Asnoted, microparticles having a diameter of between about 0.5 and about10 μm can reach the lungs, successfully passing most of the naturalbarriers. Particles in this size range can be readily prepared fromFDKP.

FDKP possesses two asymmetric centers in the diketopiperazine ring. FDKPis manufactured as a mixture of geometric isomers that are identified as“cis-FDKP” and “trans-FDKP” according to the arrangement of side chainsrelative to the central “ring” of the diketopiperazine. The R,R and S,Senantiomers have the propenyl(amidobutyl) “side arms” projecting fromthe same planar side of the diketopiperazine ring (A and B below) andare thus referred to as the cis isomers while the R,S compound has the“side arms” projecting from opposite planar sides of thediketopiperazine ring (C below) and is referred to as the trans isomer.

FDKP microparticle powders with acceptable aerodynamic performance, asmeasured by RF/fill with moderately efficient inhalers such as theMEDTONE® inhaler disclosed in U.S. Pat. No. 7,464,706 entitled, “UnitDose Cartridge and Dry Powder Inhaler,” which is incorporated byreference herein for its teachings regarding the same, have beenproduced from FDKP with a trans isomer content ranging from about 45 toabout 65%. Particles with isomer content in this range also perform wellwith high efficiency inhalers such as those disclosed in U.S. Pat. No.8,499,757 entitled, “A Dry Powder Inhaler and System for Drug Delivery,”filed on Jun. 12, 2009, U.S. Pat. No. 8,424,518 entitled “Dry PowderInhaler and System for Drug Delivery,” filed on Jun. 12, 2009, U.S.patent application Ser. No. 13/941,365 entitled “Dry Powder DrugDelivery System and Methods,” filed Jul. 12, 2013, and U.S. patentapplication Ser. No. 12/717,884, entitled, “Improved Dry Powder DrugDelivery System,” filed on Mar. 4, 2010, which disclosures are hereinincorporated by reference for their teachings regarding the same.Powders comprising microparticles containing more than 65% trans-FDKPtend to have lower and more variable RF/fill. Trans isomer-enrichedmicroparticles of FDKP have altered morphology and also lead to viscoussuspensions which are difficult to process.

Formulations of FDKP microparticles having a trans isomer content ofabout 45% to about 65% provide powders with acceptable aerodynamicproperties as disclosed in U.S. Pat. No. 8,227,409, which disclosuresare incorporated herein by reference for its teachings regarding thesame. Formulations of FDKP particles having a defined specific surfacearea less than 67 m²/g also provide dry powders for inhalation withacceptable aerodynamic properties as disclosed in U.S. Pat. No.8,551,528 entitled “Diketopiperazine Microparticles with DefinedSpecific Surface Areas” filed Jun. 11, 2010, which disclosure isincorporated herein by reference for its teachings regarding the same.These FDKP powders, however, tend to be cohesive and the inhaler isdesigned to overcome this characteristic.

It is thus desirable to produce diketopiperazine powders having aparticle composition which is less cohesive, which would allow moreeffective drug delivery and less inhaler design around. The presentdisclosure ascertains that the present method of making microcrystallineparticles of diketopiperazine as exemplified by FDKP and FDKP disodiumsalt provides microcrystalline dry powders with acceptable aerodynamicperformance which powders are less cohesive, differ in density, have analternate physical structure that does not self assemble in suspensionand provide increased capacity for drug content, including deliveringone or more active agents, which was not anticipated.

It was determined that improved consistency in homogeneity of theparticles could be obtained with a different process for making thediketopiperazine microparticles. The present methods for making thecompositions, and compositions comprising the present microcrystallinediketopiperazine particles provide dry powders for pulmonary inhalationwith beneficial physical and morphological aerodynamic characteristics.

Selection and Incorporation of Active Agents

In exemplary embodiments comprising FDKP, at least as long as themicrocrystalline particles described herein retain the above isomercontent, they can adopt other additional characteristics beneficial fordelivery to the lung and/or drug adsorption. U.S. Pat. No. 6,428,771entitled “Method for Drug Delivery to the Pulmonary System” describesDKP particle delivery to the lung and is incorporated by referenceherein for its teachings regarding the same. U.S. Pat. No. 6,444,226,entitled, “Purification and Stabilization of Peptide and ProteinPharmaceutical Agents” describes beneficial methods for adsorbing drugsonto microparticle surfaces and is also incorporated by reference hereinfor its teachings regarding the same. Microparticle surface propertiescan be manipulated to achieve desired characteristics as described inU.S. Pat. No. 7,799,344, entitled “Method of Drug Formulation based onIncreasing the Affinity of Crystalline Microparticle Surfaces for ActiveAgents,” which is incorporated by reference herein for its teachingsregarding the same. U.S. Pat. No. 7,803,404, entitled “Method of DrugFormation based on Increasing the Affinity of Active Agents forCrystalline Microparticle Surfaces” describes methods for promotingadsorption of active agents onto microparticles. U.S. Pat. No. 7,803,404is also incorporated by reference herein for its teachings regarding thesame. These teachings can be applied to the adsorption of active agentto crystallites in suspension, for example, prior to spray drying.

The microcrystalline particles described herein can comprise one or moreactive agents. As used herein “active agent”, used interchangeably with“drug”, refers to pharmaceutical substances, including small moleculepharmaceuticals, biologicals and bioactive agents. Active agents can benaturally occurring, recombinant or of synthetic origin, includingproteins, polypeptides, peptides, nucleic acids, organic macromolecules,synthetic organic compounds, polysaccharides and other sugars, fattyacids, and lipids, and antibodies and fragments thereof, including, butnot limited to, humanized or chimeric antibodies, F(ab), F(ab)₂, asingle-chain antibody alone or fused to other polypeptides ortherapeutic or diagnostic monoclonal antibodies to cancer antigens. Theactive agents can fall under a variety of biological activity andclasses, such as vasoactive agents, neuroactive agents including opioidagonist and antagonists, hormones, anticoagulants, immunomodulatingagents, cytotoxic agents, antibiotics, antiviral agents, antigens,infectious agents, inflammatory mediators, hormones, cell surfacereceptor agonist and antagonists, and cell surface antigens. Moreparticularly, active agents can include, in a non-limiting manner,cytokines, lipokines, enkephalins, alkynes, cyclosporins, anti-IL-8antibodies, IL-8 antagonists including ABX-IL-8; prostaglandinsincluding PG-12, LTB receptor blockers including LY29311, BIIL 284 andCP105696; triptans such as sumatriptan and palmitoleate, insulin andanalogs thereof, growth hormone and analogs thereof, parathyroid hormone(PTH) and analogs thereof, parathyroid hormone related peptide (PTHrP),ghrelin, obestatin, enterostatin, granulocyte macrophage colonystimulating factor (GM-CSF), amylin, amylin analogs, glucagon-likepeptide 1 (GLP-1), clopidogrel, PPACK(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), oxyntomodulin(OXM), peptide YY(3-36) (PYY), adiponectin, cholecystokinin (CCK),secretin, gastrin, glucagon, motilin, somatostatin, brain natriureticpeptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormonereleasing factor (GHRF), integrin beta-4 precursor (ITB4) receptorantagonist, analgesics, nociceptin, nocistatin, orphanin FQ2,calcitonin, CGRP, angiotensin, substance P, neurokinin A, pancreaticpolypeptide, neuropeptide Y, delta-sleep-inducing peptide and vasoactiveintestinal peptide; and analogs of the active agents.

The drug content to be delivered on microcrystalline particles formedfrom FDKP or FDKP disodium salt can typically be greater than 0.01%(w/w). In one embodiment, the drug content to be delivered with themicrocrystalline particles can be from about 0.01% (w/w) to about 75%(w/w); from about 1% to about 50% (w/w), from about 10% (w/w) to about30% (w/w), or from about 10% to about 20% (w/w). In one embodiment, forexample, if the drug is insulin, the present microparticles typicallycomprise approximately 10% to 45% (w/w), or from about 10% to about 20%(w/w) insulin. In certain embodiments, the drug content of the particlescan vary depending on the form and size of the drug to be delivered. Inan embodiment wherein GLP-1 is used as an active agent, the GLP-1content can be up to 40% (w/w) of the powder content.

In an embodiment, a composition comprising more than one active agentcan be made using the present method by adsorption, for example bybinding the active agent to the crystallites before forming the drypowder.

In an embodiment, a composition comprising more than one active agentcan be made using the present method by entrapping the active agentbetween and amongst the crystallites, for example by spray drying thematerial, without first adsorbing the active agent to the crystallites.

The method of making such composition can comprise the steps of makingmicrocrystalline diketopiperazine particles comprising more than oneactive agents; wherein each active agent/ingredient is processedseparately in a solution and added to separate suspensions ofdiketopiperazine particles, then the two or more separate suspensionscomprising the active agents are blended prior to dispersing andspray-drying the particles.

In certain embodiments, crystallites can be mixed with a solutioncomprising one or more active agents.

In certain embodiments, crystallites can be mixed with a solutioncomprising one or more active agents wherein solution conditions arechanged to promote adsorption of the active agent on to the crystallitesurface.

Each of multiple active agents can be adsorbed to a separate aliquot orspecies of crystallites. The aliquot-adsorbed crystallites can then bemixed together and spray dried. Alternatively, an aliquot can contain noactive agent so as to asjust the overall content of the active agent inthe dry powder without altering the conditions used to adsorb the activeagent on to the crystallites.

In an alternate embodiment, the one or more independent solutionscontaining a single active agent can be combined with a suspensioncomprising the diketopiperazine particles prior to dispersing andspray-drying to reform particles. The resultant dry powder compositioncomprises two or more active ingredients. In this embodiment, the amountof each ingredient can be controlled in the composition depending on theneed of the patient population to be treated.

As is evident from the foregoing disclosure, microparticles ofembodiments disclosed herein can take many different forms andincorporate many different drugs or active agents.

EXAMPLES

The following examples are included to demonstrate embodiments of thedisclosed microcrystalline diketopiperazine particles. It should beappreciated by those of skill in the art that the techniques disclosedin the examples which follow represent techniques developed by theinventors to function well in the practice of the present disclosure,and thus can be considered to constitute preferred modes for itspractice. However, those of ordinary skill in the art should, in lightof the present disclosure, appreciate that many changes can be made inthe specific embodiments which are disclosed and still obtain a like orsimilar result without departing from the scope of the invention.

Example 1

Manufacture of Standard FDKP Microparticles—A prior art manufacturingprocess was used to produce FDKP microparticles for comparison purposesas standard particles as disclosed in U.S. Pat. Nos. 7,799,344;7,803,404 and 8,227,409, which disclosures are incorporated herein byreference for their teachings of the relevant subject matter. Insummary, the typical FDKP particle formation process, feed solutions ofFDKP and acetic acid, each containing 0.05%(w/w) polysorbate 80 (PS80),are combined in a high shear mixer. Table 1 below shows the componentsfor the FDKP and insulin stock solutions.

TABLE 1 10.5% Acetic Acid Solution filtered through 0.2 μm membraneComponent wt % DI Water 89.00 GAA 10.50 10% Polysorbate 80 0.50

TABLE 2 2.5% FDKP Solution filtered through 0.2 μm membrane Component wt% DI Water 95.40 FDKP 2.50 NH₄OH 1.60 10% Polysorbate 80 0.50

A concentrated insulin stock solution can be prepared with 1 partinsulin and 9 parts about 2% wt acetic acid. The insulin stock can beadded gravimetrically to the suspension to obtain a load of about 11.4%wt. The insulin-containing suspension can be mixed at least about 15minutes, and then titrated with about 14 to about 15 wt % aqueousammonia to a pH of about 4.5 from an initial pH of about 3.5. Thesuspension can be flash-frozen in liquid nitrogen to form pellets usinga cryogranulator, for example, as disclosed in U.S. Pat. No. 8,590,320,which disclosure is incorporated herein by reference in its entirety,and lyophilized to yield the bulk insulin-loaded FDKP microparticles,which form small crystals or clusters that self-assemble into FDKPparticles with an open structure as seen in FIGS. 1A and 1B.

Samples of particles formed were studied to measure the sizedistribution of these particles in suspension and the results are shownin FIG. 2. The data in FIG. 2 show a graphic representation of theparticle size distribution measurements which are plotted in logarithmicscale as the probability density function (pdf, left y-axis) and thecumulative distribution function (cdf, right y-axis). The data show thatthe particles in suspension have a size distribution in a single peakwhich ranges from about 1.0 to about 10 μm in diameter centered on orabout 2 μm.

Manufacture of Microcrystalline FDKP Particles—a 2.5% (w/w) FDKP wasdissolved in a basic solution of aqueous ammonia (1.6% ammonia). A 10.5%(w/w) acetic acid stock solution was added in a high shear mixer(Sonolator) at an approximate pH of 2.0 under high pressure to make theparticles. Particles formed were wash in deionized water. It was foundthat diketopiperazine microparticles are not stable without the presenceof a surfactant in the solutions, however, no surfactant was added toany of the solutions or reagents in making the particles.

In these experiments, using a dual-feed high shear mixer, equal massesof about 10.5 wt % acetic acid and about 2.5 wt % FDKP solutions atabout 16° C.±about 2° C. were fed at 2000 psi through a 0.001-in²orifice to form a precipitate by homogenization. The precipitate wascollected in a deionized (DI) water reservoir of about equal mass andtemperature. The precipitate was concentrated and washed by tangentialflow filtration with deionized water. The suspension can be finallyconcentrated to about less than 5% solids, for example, from about 2 to3.5% based on the initial mass of FDKP. The concentrated suspension canbe assayed for solids content by an oven drying method. For samplescontaining the active ingredients, i.e., insulin and/or GLP-1, asuspension of FDKP from above was used to which an insulin stocksolution (insulin dissolved in 2% acetic acid was added to thesuspension while mixing, then the suspension pH was titrated withammonium hydroxide to pH 4.5±0.3. Similarly, a GLP-1 dissolved in a 2%acetic acid stock solution was added gravimetrically with stirring to anFDKP-suspension. The GLP-1 FDKP suspension was titrated to pH 4.5±0.1.Each of the insulin-FDKP suspension and GLP-1-FDKP suspension wereindependently dispersed using an external mixing 2-fluid nozzle into aNiro SD-Micro™ Spray Dryer fitted with a high efficiency cyclone.Nitrogen was used as the process gas (25 kg/h) and the atomization fluid(2.8 kg/hr). Samples were processed using two processing conditions inthe spray dryer which are listed in Table 3.

TABLE 3 feed inlet atz Sample inlet outlet atomization ΔT % solids rateflow flow % ID T (° C.) T (° C.) P (bar) (° C.) in feed (g/min) (kg/hr)(kg/hr) yield 100 130 75 3.0 55 3.58 7.13 25 2.8 88.4 103 130 75 4.0 553.31 7.39 25 2.8 83.9

For control samples, blank FDKP microcrystalline particles weremanufactured identically minus the insulin or GLP-1 loading step.

FIG. 3 shows data from the experiment above wherein the feed solutionswere free of surfactant. FIG. 3 is a graph illustrating the particlesize distribution of a particle suspension of FDKP which exhibits atypical bimodal size distribution of the particles. The particles sizesherein range from about 0.1 to about 10 μm in diameter with onepopulation of particles being centered at 0.2 μm in diameter and theother particle population centered at 2.1 μm in diameter.

Samples of the suspensions were lyophilized and not spray-dried. FIG. 4is an SEM at 2,500× magnification of lyophilized particles. As seen inFIG. 4, upon lyophilization of a similar suspension, large flake-likeparticles were formed and gave a much larger average size whenre-suspended in water as seen in FIG. 5. FIG. 5 shows the particle sizedistribution in suspension of a sample which was freeze-dried fromparticles made without the use of a surfactant. In this study, theparticle size diameter of the resuspended particles increased from about1 to about 90 μm or more.

FIG. 6 shows a typical 2,500× magnification of a scanning electronmicrograph of powder sample from a surfactant-free preparation ofmicrocrystalline FDKP particles which was formed using the presentmethod and spray-dried as described above. As seen in FIG. 6 theparticles are homogeneously spherical in structure comprising a shell ofcrystallites. When the surfactant-free suspension was spray-dried,particles with a physical diameter of approximately 4 μm were formed asshown in FIG. 6. Unlike standard FDKP particles, these particlesdissociated into particles 0.2 μm in diameter when dispersed in water asshown in FIG. 7. Therefore, it is demonstrated that surfactants have arole in particle integrity. Dispersing the particles in 0.01 Mhydrochloric acid inhibited particle dissociation as demonstrated inFIG. 8. It is possible that dissolved FDKP precipitates during spraydrying and can be deposited along the boundaries between primaryparticles and can act as cement. The FDKP “cement” dissolves in waterand the particles dissociate into the 0.2 μm primary particles; thelower solubility of FDKP in acid prevents dissolution and preservesparticle integrity.

Example 2

Manufacture of Microcrystalline FDKP Particles by Alternate ProcessUsing a Diketopiperazine Salt—Alternatively, crystallites of FDKP can beformed from feed solutions that contain surfactant. A feed solution ofFDKP was prepared by dissolving the disodium salt of FDKP (Na₂FDKP) inwater containing polysorbate 80 (PS80) as a surfactant without the useof ammonia as a reagent. A feed solution containing acetic acid (10.5%w/w), and PS80 (0.5% w/w) was also prepared. Mixing the two feedsolutions in a DUAL FEED SONOLATOR crystallized the FDKP and yielded thebimodal particle size distribution illustrated in FIG. 9. As shown inFIG. 9, approximately 26% primary crystals formed were about 0.4 μm indiameter and about 74% of the larger particles have a diameter of about2.4 μm. This suspension was processed and spray-dried to obtainparticles and observed under SEM. The SEM micrographs were taken at2,500× and 10,000× magnification and presented in FIGS. 10A and 10B.FIGS. 10A and 10B show that the particles are similar and spherical inshape, but smaller than those shown in FIG. 6 of Example 1, whichparticles were made using the FDKP as free acid. Table 4 below showssome of the physical characteristics measured for a powder made bylyophilization and a powder made by spray-drying (SD) using FDKPdisodium salt.

TABLE 4 Bulk Tap density density SSA process % RF/fill % CE g/mL g/mL(m²/g) Lyophilized 28.0 83.8 0.019 0.030 59.9 SD 62.8 88.2 0.159 0.23449.6

The data show that the powder made from spray-dried particles exhibitedhigher respirable fraction (62.8 vs. 28%), higher cartridge emptying (%CE, 88.2% vs. 83.8%), and higher bulk (0.159 g/mL) and tap (0.234 g/mL)densities than the lyophilized powder (0.019 and 0.03 g/mL,respectively).

Example 3

Manufacture of Microcrystalline FDKP Particles Containing an ActiveAgent—An active pharmaceutical ingredient (active agent) wasincorporated into the particles by adding a solution of active agent toa suspension of surfactant-free FDKP crystallites and then spray dryingthe mixture to remove solvent as described in Example 1. Controlparticles (FDKP-insulin) were also made by the standard, self-assemblymethod using PS-80 in the solutions to make powder for pulmonaryinhalation. In this study, insulin was dissolved in dilute acetic acidand added to a suspension of surfactant-free crystallites of FDKP(Samples 1 and 2 Table 5) prepared as in Example 1. The suspension wasspray-dried to obtain a dry powder containing approximately 10 wt %insulin. Samples of the powders were taken for various analysesincluding delivery through a high resistance inhaler and scanningelectron microscopy and the results are shown in Table 5. The particleswere approximately the same size as the particles without insulin(Example 1) and the morphology of the particles (FIG. 11) was the sameas those of FIG. 6. Moreover, both Samples 1 and 2 were less densepowder than the standard particles and Sample 1 particles have largerspecific surface area (SSA) than the control. The distribution ofinsulin is not known, there are no obvious depositions of insulin on theparticle surface to suggest the insulin is in the particle interior orincorporated into the particle wall.

TABLE 5 Bulk Tapped Sample % % RF % SSA Density Density ID Insulin onFill CE (m²/g) (g/cm³) (g/cm³) Control 11.4 50 85 30-50 0.1 0.18 1 11.420.3 96.4 58.66 0.020 0.031 2 22.8 17.5 77.9 38.47 0.025 0.036The data presented in Table 5, however, show that the surfactant-freepowders behave differently from the standard particles at the sameinsulin content. For example, at the same insulin content thesurfactant-free powder was released more effectively (96.4%) from theinhaler than the standard particle (85%). The increased in percentcartridge emptying (% CE) indicates that the powder has increasedflowability. The respirable fraction (% RF on Fill) was higher for thecontrol particles as the inhaler used to test the powders was designedfor the control powders.

Example 4

Manufacture of Microcrystalline FDKP Particles by Alternate ProcessUsing a Diketopiperazine Salt—In this study, FDKP disodium salt was usedto make an FDKP salt particle suspension as described in Example 2. Aninsulin solution was added to a suspension of surfactant-freemicrocrystals of FDKP prepared as in Example 2. The suspension wasspray-dried to obtain a dry powder containing approximately 10 wt %insulin. The morphology of the particles formed is shown in the SEM atFIGS. 12A and 12B at 2,500× and 10,000× magnification (respectively). Asseen in FIGS. 12A and 12B, the morphology was the same as the particleswithout insulin, showing a spherical shaped structure having a mediandiameter of the particles of 2.6 μm as shown in FIG. 13 and illustratedby the particles also ranging in diameter from about 1.0 μm to about 10μm.

Example 5

Manufacture of Microcrystalline FDKP Particles Containing More than OneActive Agent—In another embodiment, a composition comprising more thanone active agent can be made using the present method. The method ofmaking such composition comprises the steps as disclosed above for eachindividual active agent to form an active agent-FDKP suspension of eachof the active agents to be incorporated into the composition. Then, thesuspensions are combined and blended to form a mixture. Then the blendedmixture is dispersed and spray-dried as described above to make themicrocrystalline diketopiperazine particles comprising more than oneactive agent. In one exemplary study, insulin and GLP-1 combinationpowder was made.

Suspensions of FDKP crystallites prepared as in Example 1 were mixedwith solutions of various active agents (e.g., ghrelin, low molecularheparin, oxyntomodulin) and spry dried to obtain particles similar inproperties to those in Example 3.

Example 6

Manufacture of Microcrystalline FDKP Particles Containing Two ActiveAgents —A combination powder with two active agents (GLP-1 and insulin)was produced by first preparing a suspension of FDKP crystallites withinsulin and a second suspension of crystallites with GLP-1. The twosuspensions were then mixed and the combined suspension was spray-driedto obtain a dry powder containing both active agents. The crystallitesuspensions were prepared as in Example 1; after the active agents wereadded, the suspension was adjusted to pH 4.5 to promote adsorption ontothe crystallites. FIG. 14 is a plot of data illustrating the particlesize distribution of the spray-dried combination powder (1) and thecrystallite suspension with the individual active agents, FDKP-insulin(2) and FDKP-GLP-1.

As seen in FIG. 14, the particle size distribution of the combinationpowder was centered between those of the two individual suspensions andwas significantly narrower. The combination powder comprised particleshaving a diameter of from about 1 μm to about 10 μm. The crystallitescontaining the insulin were smaller (from about 0.25 μm to about 10 μm)than the GLP-1 containing crystallites which have a diameter rangingfrom about 0.5 μm to about 50 μm. The atomization step in spray dryingprobably dissociates the original clusters of crystallites in suspensionand re-forms the particles with a size distribution that depends on theconditions in the suspension and spray-drying conditions.

Example 7

Administration of a Dry Powder Composition Comprising CrystallineDiketopiperazine Particles to a Subject.

Dry powder formulations comprising microcrystalline diketopiperazinemicroparticles made with the disodium salt of FDKP (Na₂FDKP) were madeas described in Example 1 above to contain 9 U of insulin per milligramof composition. High resistance inhalers containing a cartridge(Dreamboat™ inhaler, MannKind Corporation) were prepared containing 1 mgto 10 mg per dose were prepared to administer to subjects diagnosed withdiabetes. An inhaler containing an insulin dose is provided to thepatient to be treated and the patient inhales the insulin dose in asingle inhalation at the start, during a meal or thereafter a meal.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

Further, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed is:
 1. A crystalline diketopiperazine compositioncomprising a plurality of microcrystalline particles comprising anactive agent, said microcrystalline particles being substantiallyuniform in size and having a volumetric median geometric diameter lessthan 5 μm, and further having hollow spherical structures; each of saidhollow spherical structures comprising a core and a shell, wherein saidshell is porous and comprises crystallites of the diketopiperazine, saiddiketopiperazine having the formula:

and a trans isomer content ranging from 45% to 65%, wherein saidmicrocrystalline particles dissolve into crystallites when dispersed inwater; wherein said microcrystalline particles are prepared in asurfactant-free solution, and said microcrystalline particles do notself-assemble in said solution, wherein the microcrystalline particlesare formed by a method comprising the steps of; combining3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine dissolved in asolution of aqueous ammonia with a solution of acetic acid andconcurrently homogenizing the solutions in a high shear mixer at highpressure of up to 2000 psi to form a precipitate; washing theprecipitate in suspension with deionized water; concentrating thesuspension; and drying the suspension in a spray drying apparatus. 2.The crystalline diketopiperazine composition of claim 1, wherein up to92% of the microcrystalline particles have a volumetric median geometricdiameter of <5.8 μm.
 3. The crystalline diketopiperazine composition ofclaim 1, wherein the one or more active ingredients is a peptide, aprotein, a nucleic acid molecule, or a small organic molecule.
 4. Thecrystalline diketopiperazine composition of claim 3, wherein the peptideis an endocrine hormone.
 5. The crystalline diketopiperazine compositionof claim 4, wherein the endocrine hormone is insulin, parathyroidhormone, calcitonin, glucagon, glucagon-like peptide 1, oxyntomodulin,peptide YY, leptin, or an analog of said endocrine hormone.
 6. A methodof making microcrystalline diketopiperazine particles of claim 1suitable for pulmonary administration as a dry powder comprising: a)forming diketopiperazine particles in a suspension having a bimodaldistribution in the particle sizes which range from about 0.05 pm toabout 10 pm; b) atomizing the suspension using a spray dryer under anair or gas stream, and c) reforming particles by spray-drying into a drypowder comprising the microcrystalline diketopiperazine particles havinghollow spheres.
 7. The method of claim 6 wherein a first peak ofparticles of the bimodal distribution in particle sizes has an averageof about 0.2 pm to about 2.4 pm and a second peak of particles has anaverage size of about 2.1 pm to about 2.4 pm.
 8. The method of claim 6,wherein the diketopiperazine is of the formula3,6-bis(N-X-4-aminobutyl)-2,5-diketopiperazine, wherein X is selectedfrom the group consisting of fumaryl, succinyl, maleyl, malonyl, andglutaryl.
 9. The method of claim 8, wherein the diketopiperazine is3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine.
 10. The method ofclaim 6, further comprising the step of adding a solution comprising oneor more active agents to the suspension in step a).
 11. The method ofclaim 6, further comprising the step of adding a surfactant to thesolution or suspension.
 12. The method of claim 11, wherein thesurfactant is polysorbate
 80. 13. The method of claim 10, wherein theone or more active agent is a peptide, a protein, a nucleic acidmolecule, or a small organic molecule.
 14. The method of claim 13,wherein the peptide is an endocrine hormone.
 15. The method of claim 14,wherein the endocrine hormone is insulin, parathyroid hormone,calcitonin, glucagon, glucagon-like peptide 1, oxyntomodulin, peptideYY, leptin, or an analog of said endocrine hormone.
 16. The crystallinediketopiperazine composition of claim 1, wherein said active agentcomprises at least one of small molecule pharmaceuticals, biologicals,and bioactive agents.
 17. The crystalline diketopiperazine compositionof claim 1, wherein said active agent comprises at least one ofproteins, polypeptides, peptides, nucleic acids, organic macromolecules,synthetic organic compounds, polysaccharides and other sugars, fattyacids, and lipids, and antibodies and fragments thereof.
 18. Thecrystalline diketopiperazine composition of claim 1, wherein said agentcomprises at least one of humanized or chimeric antibodies, F(ab),F(ab)₂, a single- chain antibody alone or fused to other polypeptides ortherapeutic or diagnostic monoclonal antibodies to cancer antigens,vasoactive agents, neuroactive agents, hormones, anticoagulants,immunomodulating agents, cytotoxic agents, antibiotics, antiviralagents, antigens, infectious agents, inflammatory mediators, hormones,cell surface receptor agonists and antagonists, and cell surfaceantigens.
 19. The crystalline diketopiperazine composition of claim 1,wherein said agent comprises at least one of cytokines, lipokines,enkephalins, alkynes, cyclosporins, anti-IL-8 antibodies, IL-8antagonists including ABX-IL-8; PG-i2, LTB receptor blockers; triptans,insulin and analogs thereof, growth hormone and analogs thereof,parathyroid hormone (PTH) and analogs thereof, parathyroid hormonerelated peptide (PTHrP), ghrelin, obestatin, enterostatin, granulocytemacrophage colony stimulating factor (GM-CSF), amylin, amylin analogs,glucagon-like peptide 1 (GLP-1), clopidogrel, PPACK(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), oxyntomodulin(OXM), peptide YY(3-36) (PYY), adiponectin, cholecystokinin (CCK),secretin, gastrin, glucagon, motilin, somatostatin, brain natriureticpeptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormonereleasing factor (GHRF), integrin beta-4 precursor (ITB4) receptorantagonist, analgesics, nociceptin, nocistatin, orphanin FQ2,calcitonin, CGRP, angiotensin, substance P, neurokinin A, pancreaticpolypeptide, neuropeptide Y, delta-sleep-inducing peptide, vasoactiveintestinal peptide; and analogs of the active agents.
 20. Thecrystalline diketopiperazine composition of claim 1, wherein said agentcomprises a prostaglandin.
 21. The crystalline diketopiperazinecomposition of claim 20, wherein said prostaglandin comprises PG-I2.